Load libraries and working directory

Dimer check - Use sample_coverage files

## # A tibble: 15 × 2
##    run   dimer_percent
##    <chr>         <dbl>
##  1 M03            38.7
##  2 M04            22.4
##  3 M05            33.0
##  4 M06            60.7
##  5 M07            29.7
##  6 M08            24.5
##  7 M09            23.5
##  8 M10            25.0
##  9 M11            23.5
## 10 M12            26.3
## 11 M13            19.7
## 12 M14            11.3
## 13 M15            47.8
## 14 NS1            12.4
## 15 NS2            45.8

Preview data structure

Merge allele, gene (amplicon info), and meta data

Preview raw data - sample size, loci numbers, allele numbers

## [1] "Sample size= 1630"
## [1] "All locus = 274"
## [1] "1 to 10 alleles per locus per sample"
## [1] "Mean of total loci counts = 219.46, Mean prop. of monoclonal loci = 0.97, Mean prop. of polyclonal loci = 0.03"

Preview raw data - Plate map, Detect contamination and Check balanced

Check negative controls

## # A tibble: 17 × 1
##    sampleID  
##    <chr>     
##  1 M09_NC3   
##  2 M13_NC3   
##  3 M13_NC4   
##  4 M13_NC7   
##  5 M13_NC8   
##  6 M15_NC3   
##  7 M15_NC6   
##  8 M13_NC1   
##  9 M13_NC2   
## 10 M13_NC6   
## 11 M10_NC7   
## 12 M13_NC5   
## 13 M15_NC1   
## 14 M15_NC4   
## 15 M15_NC2   
## 16 M07_NC-D05
## 17 M15_NC5

Check positive controls

## # A tibble: 32 × 2
##    sampleID   pc_dup_prop
##    <chr>            <dbl>
##  1 M07_PC-G04       0.867
##  2 M08_PC1          0.992
##  3 M08_PC2          0.996
##  4 M09_PC1          0.996
##  5 M09_PC2          0.987
##  6 M10_PC1          0.996
##  7 M10_PC2          0.987
##  8 M10_PC3          0.996
##  9 M11_PC1          0.987
## 10 M11_PC2          0.987
## # ℹ 22 more rows

Remove contaminated runs

## [1] "Sample size= 1239 Retained prop. = 0.76"
## [1] "All locus = 274"
## [1] "1 to 6 alleles per locus per sample"

Data QC and filtering

  1. Filter out bad-quality samples and possible false positive alleles (off-targets, low-abundance)
  2. Make moire input file

Step 1. Total reads

## [1] "Step 1 - Sample size after removing samples with insufficient reads = 1043, Retained prop. = 0.84"
##   [1] "MH-05-AC_HAM14-000168"  "MH-13-AC_HT29052011-3"  "M14_HAM14-066-D0"      
##   [4] "M14_HT07032012-05"      "MH-02-AC_HAM15-000700"  "MH-02-AC_HAM15-000780" 
##   [7] "MH-02-AC_HAM15-000669"  "MH-02-AC_HAM15-000734"  "MH-02-AC_HAM15-000712" 
##  [10] "M14_HT20022011-01"      "MH-02-AC_HAM15-000696"  "MH-03-AC_HAM15-000636" 
##  [13] "MH-03-AC_HAM15-000750"  "M14_HT05012011-03"      "M14_HT11022013-07"     
##  [16] "M05_HAM16-185-D0"       "MH-03-AC_HAM15-000210"  "M14_HAM15-037-D0"      
##  [19] "M05_HAM16-169-D0"       "M05_HAM16-111-D0"       "M14_HT14122012-11"     
##  [22] "M05_HAM16-303-D0"       "M05_HAM16-373-D0"       "M12_HT23012013-01"     
##  [25] "M05_HAM16-182-D0"       "MH-03-AC_HAM15-000561"  "MH-11-AC_HT29072010-1" 
##  [28] "M05_HAM16-157-D0"       "M05_HAM16-358-D0"       "MH-02-AC_HAM15-000707" 
##  [31] "M12_HT19112012-01"      "M14_HT11012013-07"      "M12_HT17072012-07"     
##  [34] "M12_HT14122012-11"      "M12_HT11012013-05"      "MH-13-AC_HT08042011-3" 
##  [37] "MH-05-AC_HT02112013-1"  "M12_HT22122012-07"      "M12_HT14012013-05"     
##  [40] "MH-13-AC_HT13032011-4"  "M12_HT02122012-01"      "M12_HT11012013-03"     
##  [43] "M14_HT28062010-01"      "MH-11-AC_HT07062010-2"  "MH-12-AC_HT15022011-1" 
##  [46] "MH-03-AC_2007-15"       "MH-03-AC_2007-3a"       "MH-05-AC_HAM14-000163" 
##  [49] "MH-03-AC_2007-5a"       "MH-03-AC_2007-7a"       "MH-13-AC_HT09122010-2" 
##  [52] "MH-03-AC_2007-32"       "MH-03-AC_2007-1a"       "M03_HAM16-123-D0"      
##  [55] "M12_HT02022012-02"      "MH-05-AC_HT01062013-7"  "MH-13-AC_HT06112010-2" 
##  [58] "M12_HT21092012-03"      "MH-11-AC_HT12042010-5"  "MH-13-AC_HT18052011-21"
##  [61] "MH-05-AC_HT27112013-6"  "MH-05-AC_HT12072013-4"  "MH-13-AC_HT23052011-2" 
##  [64] "MH-13-AC_HT28022011-1"  "MH-11-AC_HT17062010-4"  "MH-05-AC_HT03052013-11"
##  [67] "MH-03-AC_2005-121"      "MH-12-AC_HT10072010-1"  "MH-12-AC_HT15042011-5" 
##  [70] "M03_HT11012013-03"      "MH-09-AC_HT19112012-4"  "MH-04-AC_HAM14-000378" 
##  [73] "M14_HAM14-128-D0"       "M08_HAM15-266-D0"       "MH-05-AC_HT02072013-14"
##  [76] "M12_HT07072012-01"      "MH-13-AC_HT28042011-1"  "MH-04-AC_HAM14-000517" 
##  [79] "MH-12-AC_HT14062010-3"  "M14_HT20072012-03"      "M12_HT20072012-03"     
##  [82] "MH-04-AC_HAM14-000337"  "MH-12-AC_HT06082010-1"  "MH-06-AC_HT01072013-8" 
##  [85] "M14_HAM14-052-D0"       "MH-06-AC_HT09022013-1"  "M14_HAM14-141-D0"      
##  [88] "M14_HAM15-302-D0"       "M08_HAM16-110-D0"       "MH-12-AC_HT23072010-2" 
##  [91] "M14_HAM16-199-D0"       "MH-05-AC_HAM14-000581"  "M12_HAM16-199-D0"      
##  [94] "M14_HT14012013-05"      "MH-05-AC_HAM14-000459"  "MH-12-AC_HT18012011-2" 
##  [97] "M06_HAM16-260-D0"       "MH-05-AC_HT31052013-6"  "MH-12-AC_HT14122010-5" 
## [100] "MH-12-AC_HT27122010-3"  "MH-12-AC_HT04032011-2"  "MH-04-AC_HAM14-000514" 
## [103] "MH-13-AC_HT12122011-4"  "M08_HAM15-122-D0"       "MH-12-AC_HT20022011-2" 
## [106] "M14_HAM14-108-D0"       "MH-08-AC_HT13112012-5"  "MH-05-AC_HAM14-000392" 
## [109] "MH-12-AC_HT15112010-1"  "M12_HT21072012-02"      "MH-05-AC_HAM14-000233"

Step 2. Loci coverage

## [1] "Step 2 - Sample size after removing samples with insufficeint loci coverage = 1022, Retained prop. = 0.98"
##  [1] "M11_HT16032010-05"     "MH-04-AC_HAM14-000464" "MH-11-AC_HT09052010-3"
##  [4] "MH-11-AC_HT26052010-1" "MH-12-AC_HT02112010-1" "MH-12-AC_HT02112010-2"
##  [7] "MH-12-AC_HT05082010-1" "MH-12-AC_HT08052011-2" "MH-12-AC_HT13072010-1"
## [10] "MH-12-AC_HT15072010-2" "MH-12-AC_HT15122010-1" "MH-12-AC_HT16032011-1"
## [13] "MH-12-AC_HT16122010-1" "MH-12-AC_HT19112010-1" "MH-12-AC_HT20092010-2"
## [16] "MH-12-AC_HT20102010-3" "MH-12-AC_HT21052010-2" "MH-12-AC_HT24112010-1"
## [19] "MH-12-AC_HT27042011-3" "MH-13-AC_HT25072011-1" "MH-13-AC_HT28022011-5"

Step 3: Proportion of loci > 100 reads in samples

## [1] "Step 3 - Sample size after removing samples with less than n100 cutoff = 992, Retained prop. = 0.97"
##  [1] "M06_HAM16-016-D0"       "M06_HAM16-290-D0"       "M08_HAM16-111-D0"      
##  [4] "M12_HAM15-290-D0"       "M14_HAM15-623-D0"       "MH-02-AC_HAM15-000628" 
##  [7] "MH-02-AC_HAM15-000754"  "MH-05-AC_HT05102013-2"  "MH-05-AC_HT13072013-3" 
## [10] "MH-05-AC_HT15052013-1"  "MH-05-AC_HT23062013-1"  "MH-06-AC_HT24042013-5" 
## [13] "MH-06-AC_HT29072013-1"  "MH-06-AC_HT29092013-2"  "MH-08-AC_HT03082012-4" 
## [16] "MH-08-AC_HT04062012-6"  "MH-08-AC_HT04122012-12" "MH-08-AC_HT14072012-1" 
## [19] "MH-08-AC_HT16062012-11" "MH-08-AC_HT17112012-4"  "MH-08-AC_HT22122012-3" 
## [22] "MH-08-AC_HT24082012-2"  "MH-12-AC_HT11112010-1"  "MH-12-AC_HT14122010-7" 
## [25] "MH-12-AC_HT18042011-8"  "MH-12-AC_HT29122010-1"  "MH-13-AC_HT05022011-2" 
## [28] "MH-13-AC_HT08032011-5"  "MH-13-AC_HT23032012-4"  "MH-13-AC_HT29042011-8"

Check if there are any loci not amplified well, and if this happened in one sample or many samples

## [1] "126 loci had <= 10 reads at least in one sample"
## [1] "2 loci had <= 10 reads across 1/3 of total samples"
## [1] "Which loci had <= 10 reads across 1/3 of total samples?"
## [1] "Pf3D7_03_v3-240957-241256-2"  "Pf3D7_09_v3-685560-685825-1A"

qPCR density and sample info

Optional - remove minor alleles

## [1] "Percentage (x100) of excluded minor alleles in total alleles = 3.088, Percentage (x100) of excluded minor alleles in total data entries = 0.078 (x100)"
## [1] "Percentage (x100) of excluded minor alleles in total alleles = 3.088, Percentage (x100) of excluded minor alleles in total data entries = 0.078 (x100)"
##   [1] "t10.108"  "t10.109"  "t10.128"  "t10.13"   "t10.150"  "t10.155" 
##   [7] "t10.164"  "t10.25"   "t10.29"   "t10.65"   "t10.67"   "t10.70"  
##  [13] "t10.73"   "t10.76"   "t108.3"   "t111.19"  "t140.11"  "t140.12" 
##  [19] "t140.13"  "t140.16"  "t140.48"  "t140.53"  "t140.55"  "t140.58" 
##  [25] "t141.27"  "t154.17"  "t154.49"  "t154.52"  "t154.6"   "t155.125"
##  [31] "t155.66"  "t156.17"  "t156.43"  "t156.65"  "t16.11"   "t162.27" 
##  [37] "t164.16"  "t167.19"  "t17.55"   "t17.59"   "t17.68"   "t171.39" 
##  [43] "t171.48"  "t181.10"  "t19.4"    "t192.36"  "t193.103" "t193.16" 
##  [49] "t193.29"  "t193.3"   "t193.31"  "t193.44"  "t194.12"  "t194.20" 
##  [55] "t194.43"  "t194.44"  "t194.9"   "t196.26"  "t2.24"    "t200.19" 
##  [61] "t207.23"  "t207.25"  "t207.30"  "t21.2"    "t21.33"   "t217.3"  
##  [67] "t217.33"  "t217.35"  "t219.52"  "t223.3"   "t223.50"  "t223.66" 
##  [73] "t231.18"  "t231.4"   "t239.8"   "t245.5"   "t246.23"  "t250.8"  
##  [79] "t258.18"  "t262.39"  "t266.19"  "t266.43"  "t266.49"  "t266.53" 
##  [85] "t272.35"  "t4.14"    "t4.16"    "t4.24"    "t4.41"    "t4.44"   
##  [91] "t4.45"    "t4.47"    "t4.48"    "t4.49"    "t4.50"    "t4.52"   
##  [97] "t4.55"    "t4.56"    "t4.58"    "t4.61"    "t40.18"   "t40.21"  
## [103] "t49.21"   "t49.3"    "t6.39"    "t6.40"    "t60.28"   "t85.3"   
## [109] "t90.20"

Make moire input

Moire input